xl-888 and Melanoma

Novel therapies are undergoing clinical trials, for example, the Hsp90 inhibitor, XL888, in combination with BRAF inhibitors for the treatment of therapy-resistant melanomas. Unfortunately, our data show that this combination elicits a heterogeneous response in a panel of melanoma cell lines including PDX-derived models. We sought to understand the mechanisms underlying the differential responses and suggest a patient stratification strategy. Thermal proteome profiling (TPP) identified the protein targets of XL888 in a pair of sensitive and unresponsive cell lines. Unbiased proteomics and phosphoproteomics analyses identified CDK2 as a driver of resistance to both BRAF and Hsp90 inhibitors and its expression is regulated by the transcription factor MITF upon XL888 treatment. The CDK2 inhibitor, dinaciclib, attenuated resistance to both classes of inhibitors and combinations thereof. Notably, we found that MITF expression correlates with CDK2 upregulation in patients; thus, dinaciclib would warrant consideration for treatment of patients unresponsive to BRAF-MEK and/or Hsp90 inhibitors and/or harboring MITF amplification/overexpression.

Topics: Azabicyclo Compounds; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Cell Survival; Cyclic N-Oxides; Cyclin-Dependent Kinase 2; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; HSP90 Heat-Shock Proteins; Humans; Imidazoles; Indolizines; Melanoma; Microphthalmia-Associated Transcription Factor; Oximes; Phosphoproteins; Phthalic Acids; Proteomics; Pyridinium Compounds; Up-Regulation

Topics: Azabicyclo Compounds; Biopsy, Needle; Cell Proliferation; Drug Interactions; Humans; Immunohistochemistry; Indoles; Melanoma; Mitogen-Activated Protein Kinase Kinases; Phthalic Acids; Sensitivity and Specificity; Skin; Skin Neoplasms; Sulfonamides; Tissue Culture Techniques; Vemurafenib

The evolution of cancer therapy into complex regimens with multiple drugs requires novel approaches for the development and evaluation of companion biomarkers. Liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) is a versatile platform for biomarker measurement. In this study, we describe the development and use of the LC-MRM platform to study the adaptive signaling responses of melanoma cells to inhibitors of HSP90 (XL888) and MEK (AZD6244). XL888 had good anti-tumor activity against NRAS mutant melanoma cell lines as well as BRAF mutant cells with acquired resistance to BRAF inhibitors both in vitro and in vivo. LC-MRM analysis showed HSP90 inhibition to be associated with decreased expression of multiple receptor tyrosine kinases, modules in the PI3K/AKT/mammalian target of rapamycin pathway, and the MAPK/CDK4 signaling axis in NRAS mutant melanoma cell lines and the inhibition of PI3K/AKT signaling in BRAF mutant melanoma xenografts with acquired vemurafenib resistance. The LC-MRM approach targeting more than 80 cancer signaling proteins was highly sensitive and could be applied to fine needle aspirates from xenografts and clinical melanoma specimens (using 50 μg of total protein). We further showed MEK inhibition to be associated with signaling through the NFκB and WNT signaling pathways, as well as increased receptor tyrosine kinase expression and activation. Validation studies identified PDGF receptor β signaling as a potential escape mechanism from MEK inhibition, which could be overcome through combined use of AZD6244 and the PDGF receptor inhibitor, crenolanib. Together, our studies show LC-MRM to have unique value as a platform for the systems level understanding of the molecular mechanisms of drug response and therapeutic escape. This work provides the proof-of-principle for the future development of LC-MRM assays for monitoring drug responses in the clinic.

Topics: Animals; Azabicyclo Compounds; Benzimidazoles; beta Catenin; Biomarkers, Tumor; Cell Line, Tumor; Chromatography, High Pressure Liquid; Drug Resistance, Neoplasm; GTP Phosphohydrolases; HSP90 Heat-Shock Proteins; Humans; Indoles; MAP Kinase Kinase 1; MAP Kinase Signaling System; Mass Spectrometry; Melanoma; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, SCID; Neoplasm Transplantation; NF-kappa B; Phosphoinositide-3 Kinase Inhibitors; Phthalic Acids; Piperidines; Proteomics; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-akt; Receptor, Platelet-Derived Growth Factor beta; RNA Interference; RNA, Small Interfering; Sulfonamides; Transplantation, Heterologous; Vemurafenib; Wnt Signaling Pathway

The HSP90 inhibitor XL888 is effective at reversing BRAF inhibitor resistance in melanoma, including that mediated through acquired NRAS mutations. The present study has investigated the mechanism of action of XL888 in NRAS-mutant melanoma. Treatment of NRAS-mutant melanoma cell lines with XL888 led to an inhibition of growth, G2-M phase cell-cycle arrest, and the inhibition of cell survival in three-dimensional spheroid and colony formation assays. In vitro, HSP90 inhibition led to the degradation of ARAF, CRAF, Wee1, Chk1, and cdc2 and was associated with decreased mitogen-activated protein kinase (MAPK), AKT, mTOR, and c-jun NH2 kinase (JNK) signaling. Apoptosis induction was associated with increased BIM expression and a decrease in the expression of the prosurvival protein Mcl-1. The critical role of increased BIM and decreased Mcl-1 expression in the survival of NRAS-mutant melanoma cell lines was shown through siRNA knockdown and overexpression studies. In an animal xenograft model of NRAS-mutant melanoma, XL888 treatment led to reduced tumor growth and apoptosis induction. Important differences in the pattern of client degradation were noted between the in vivo and in vitro studies. In vivo, XL888 treatment led to degradation of CDK4 and Wee1 and the inhibition of AKT/S6 signaling with little or no effect observed upon ARAF, CRAF, or MAPK. Blockade of Wee1, using either siRNA knockdown or the inhibitor MK1775, was associated with significant levels of growth inhibition and apoptosis induction. Together, these studies have identified Wee1 as a key target of XL888, suggesting novel therapeutic strategies for NRAS-mutant melanoma.

Topics: Animals; Apoptosis; Azabicyclo Compounds; Cell Cycle Proteins; Cell Line, Tumor; Cyclin-Dependent Kinase 4; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; GTP Phosphohydrolases; HSP90 Heat-Shock Proteins; Humans; Melanoma; Membrane Proteins; Mice; Molecular Targeted Therapy; Nuclear Proteins; Oncogene Protein v-akt; Phthalic Acids; Protein-Tyrosine Kinases; RNA, Small Interfering; Signal Transduction; Xenograft Model Antitumor Assays

The clinical use of BRAF inhibitors is being hampered by the acquisition of drug resistance. This study shows the potential therapeutic use of the HSP90 inhibitor (XL888) in six different models of vemurafenib resistance.. The ability of XL888 to inhibit growth and to induce apoptosis and tumor regression of vemurafenib-resistant melanoma cell lines was shown in vitro and in vivo. A novel mass spectrometry-based pharmacodynamic assay was developed to measure intratumoral HSP70 levels following HSP90 inhibition in melanoma cell lines, xenografts, and melanoma biopsies. Mechanistic studies were carried out to determine the mechanism of XL888-induced apoptosis.. XL888 potently inhibited cell growth, induced apoptosis, and prevented the growth of vemurafenib-resistant melanoma cell lines in 3-dimensional cell culture, long-term colony formation assays, and human melanoma mouse xenografts. The reversal of the resistance phenotype was associated with the degradation of PDGFRβ, COT, IGFR1, CRAF, ARAF, S6, cyclin D1, and AKT, which in turn led to the nuclear accumulation of FOXO3a, an increase in BIM (Bcl-2 interacting mediator of cell death) expression, and the downregulation of Mcl-1. In most resistance models, XL888 treatment increased BIM expression, decreased Mcl-1 expression, and induced apoptosis more effectively than dual mitogen-activated protein-extracellular signal-regulated kinase/phosphoinositide 3-kinase (MEK/PI3K) inhibition.. HSP90 inhibition may be a highly effective strategy at managing the diverse array of resistance mechanisms being reported to BRAF inhibitors and appears to be more effective at restoring BIM expression and downregulating Mcl-1 expression than combined MEK/PI3K inhibitor therapy.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Azabicyclo Compounds; Bcl-2-Like Protein 11; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Colony-Forming Units Assay; Drug Resistance, Neoplasm; Extracellular Signal-Regulated MAP Kinases; Flow Cytometry; Fluorescent Antibody Technique; Forkhead Box Protein O3; Forkhead Transcription Factors; HSP90 Heat-Shock Proteins; Humans; Immunoenzyme Techniques; Indoles; Melanoma; Membrane Proteins; Mice; Mice, Inbred BALB C; Myeloid Cell Leukemia Sequence 1 Protein; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phthalic Acids; Prospective Studies; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sulfonamides; Vemurafenib

The RAF inhibitor vemurafenib has unprecedented activity in BRAF-mutant melanomas, but resistance invariably develops. As Hsp90 is required for the stability of several of the oncoproteins that mediate RAF inhibitor resistance, inhibitors of this cellular chaperone may be effective in patients with intrinsic or acquired resistance to RAF inhibitors.

Topics: Animals; Azabicyclo Compounds; Drug Resistance, Neoplasm; HSP90 Heat-Shock Proteins; Humans; Melanoma; Phthalic Acids; Proto-Oncogene Proteins B-raf; Signal Transduction